The cellular slime mold Dictyostelium discoideum is one of the simplest organisms to undergo true multicellular differentiation. Its relative simplicity offers the opportunity to study the interplay between receptor occupancy, signal transduction and spatially localized patterns of gene expression during development. The particular group of genes being studied, the prespore genes, are regulated at the transcriptional level in a temporal and spatial manner by cAMP - cell surface receptor occupancy. Additionally, the ability to stabilize the mRNA transcripts from these genes depends upon aspects of the multicellular environment. When developing cells are removed from their multicellular environment by disaggregation the prespore mRNAs are rapidly and specifically degraded. In this proposal attention is being focused on defining two aspects of the cis-acting regulation which places two members of this gene family under the above controls. In the promoter region elements important for correct spatial, temporal and cAMP mediated regulation of gene transcription will be defined. In the mRNAs work will be directed towards identifying the sites that render these mRNAs targets for disaggregation induced destabilization. A major question in development has been whether cells differentiate as a function of their position or whether they differentiate in a cell autonomous manner and then sort to position. Using reporter gene constructs fused to prespore gene promoters it has become clear that randomly located cells initiate the expression of prespore genes and then sort to their position in the posterior of the developing slugs. Comparison of the sequences of several different prespore gene promoters reveals considerable variation in potential regulatory sites. This raises the question of whether these prespore genes which appear to be coordinately induced by cAMP are if fact coordinately regulated at the individual cell level. Immunofluorescence microscopy with double labeled antibody probes will be used to determine if there is coordinate activation of expression of different members of the prespore gene family in the randomly isolated single cells that have started to express prespore genes in the early mound stage of development.